The development of simple, cost-effective, sensitive, and specific point-of-care (POC) diagnostics represents a major challenge in the 21st century. [1] The spread of infectious diseases has caused significant losses in global economy and life. A method to control the spread of diseases is to detect the pathogens early at POC, and to administer proper treatment or quarantine. [2] Detection of DNA as biomarkers is currently a widely used technique for pathogenic detection. [3] Quantitative PCR (qPCR) is the method of choice, since it includes an enzymatic signal amplification step to achieve highly sensitive and specific detection of genetic targets. [4] However, qPCR requires expensive equipment and highly trained personnel, limiting the technique use to medical laboratories. There is a strong need to develop cheaper and simpler methods for detecting DNA targets and/or other biological targets. [5]
DNAzyme is a synthetic DNA enzyme that can catalyze the cleavage of another nucleic acid molecule. [6-8] Since the catalysis is carried out with multiple turnover, DNAzyme introduces an enzymatic amplification step into the experimental setup. [9] This amplification is performed without the need for protein components, which are costly and have low thermal and storage stability. While initially designed to detect Pb2+ and other divalent cations, [10-19] a number of modifications have been reported that allow DNAzyme to detect biomolecular targets. [9,20-27] Two main approaches were used to implement detection of genetic targets. The first method utilizes competitive activation of peroxidase-mimicking DNAzyme, which can catalyze production of colorimetric or chemiluminescent product. [28] In the second, more sensitive approach, a similar strategy was followed by a number of groups to implement detection of genetic targets by splitting the DNAzyme into inactive components, which could be reactivated by the target binding. [9, 23-25, 29] However, these studies used fluorescence as the readout, which is not an optimal detection modality for POC applications since it requires access to fairly complex fluorometer apparatus. An alternative method using the colorimetric readout of gold nanoparticles (GNPs) has been reported for detection of metal ions, adenosine and cocaine. [10-14, 16-18, 20, 21] The wavelength at which GNPs absorb light is dependent on whether they are in a monodispersed or aggregated state. [30] Since the GNP solutions in monodispersed or aggregated states are easily distinguishable by their respective red or purple colors, this approach provides clear colorimetric results that can be visualized by the naked eye. [31]
As such, an object of the invention is to overcome the above limitations by combining DNAzyme technology with GNPs to engineer a simple point-of-care diagnostic platform that can be used in remote settings. [32, 33]
Further and other objects of the invention will be realized from the following Summary of the Invention, the Discussion of the Invention and the embodiments and Examples thereof.